LC-MS CONTAMINATION

General Discussion Chemical contamination is one of the more common problems with LC/MS. Therefore, please use the highest purity chemicals available. Try Burdick and Jackson solvents and water. These seem to be uniformly better than other suppliers. Switch to this brand if you have contamination problems. Additionally, use high purity acetic acid: Aldrich p/n 38,012-1 double distilled in Teflon bottle $175 for 500 Ml. Chemical contamination problems (with specific mass peaks) are rarely attributed to problems deep inside the MS. Prioritize like this: 1. HPLC pre or trap column 2. HPLC column 3. HPLC hardware 4. Chemicals 5. ESI or APCI probe 6. Spray shield area 7. Ion Transfer Tube 8. Tube lens/Skimmer 9. Q00/Lens 0/Q0 10. Analyzer Quads 11. MS Dynode Generally, if 8 11 are dirty you will see random peaks not specific masses. Identifying the source of contamination 1. Is the contamination always there or does it elute as specific peaks during the HPLC run? If the later, the problem is most likely with the HPLC. 2. Turn off flow. If the noise disappears take the column out of the flow path and test again. If the noise is still there try these items, go to step #3. 3. If you suspect that there is chemical contamination from the HPLC mobile-phase, use a clean syringe and tubing to infuse the mobilephase components individually into the MS (e.g. water, then water + acid, then methanol or acetonitrile. Generally, the acid is the source of contamination.

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LC-MS CONTAMINATION
HPLC Troubleshooting Many times contaminant peaks occur during a gradient. Try this procedure to locate the source of the contamination: Inject a solvent blank. Make a zero volume injection Run a gradient with the injection valve or autosampler out of the liquid flow.

Contamination in the MS If the noise disappears when HPLC flow is turned off and you have eliminated contamination in the mobile phase: Trim or replace the sample tube and set it to the correct distance within the needle. Clean the ESI spray nozzle (cone and needle). Replace the Teflon needle seal behind the needle If still noisy, the cause could be neutral chemical noise (non-ionized materials). Change source conditions (e.g. increase heated capillary temperature 10 or 20C, increase Sheath gas flow 10 or 20 units, increase Auxiliary gas flow 10 units.

Other possibilities Solvent reservoir pickup filters are common sources of contamination. Usually the aqueous phase one will be the first to go. Offline solvent filters are common sources of contamination. Serious contamination has been seen with the Nylon 0.22 m filters used for buffer solutions. Address the containers used for storing your acetonitrile. A similar problem occurs when using glass scintillation vials with polypropylene insert caps to makeup solvent.

Phthalate Contamination 391 413 798 803 protonated diisooctyl phthalate [M+H]+ sodium adduct of diisooctyl phthalate [M+Na]+ ammoniated dimer of diisooctyl phthalate [2M+NH4]+ sodiated dimer of diisooctyl phthalate [2M+Na]+

This is usually from contaminated solvents. It can concentrate on the column and elute during a gradient. Follow the normal solvent contamination checking procedures.

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LC-MS CONTAMINATION
Glassware cleaned by means of a dishwasher often picks up phthalate contamination. Remove this with a rinse of 30% nitric acid, followed by a rinse with 2M NH4OH. Please keep in mind, that the APCI probe can retain this. However, baking the APCI will eliminate this problem. Try 600C for 15 min. +44 Series Ions Possible Polymer Contamination If you have analyzed detergent containing samples on the system, that could explain the +44 series background problem. Also, PEGs and other ethoxylated polymers give +44 ion series. The PEG's could originate from the water, or extracted polymers from plastic ware and/or silicon coatings. Background Ramps Up With Gradient Background can originate from contaminated organic phase or the high organic content maybe eluting retained materials from the column. Substitute with Burdick and Jackson organic phase and a new column. +59 Series Ions 59 Da is the mass of the acetate ion (observed when employing ammonium acetate or acetic acid). Is it possible to have acetate polymerization? There is another explanation for the +59 ion series. Polymers of +59 might be iron in some form, presumably leaching from the steel in acid. +74 Series Ions Peaks at 536, 610 and 684 Da have been seen with LCQ DecaXP and LCQ Advantage and TSQ Quantum. The contamination came from connecting the opaque silicon tube to the API source housing. The proper tubing is Tygon. The instrument parts kits contain both types of tubing. The TSQ Quantum contains a 12-foot length of clear Tygon tube and a 2.5-foot length of opaque silicon tube. The intended function of the silicon tube is to connect from the waste container (P/N 00301-57020) to the fume exhaust system. If the silicon tube is instead connected to the API source housing, background ions at m/z 536, 610, and 684 might be observed. Solution: Remove all silicon tubing from the solvent waste system. +77 Series Ions +77 Da clusters, mostly across the mass range, can be attributed to the iontransfer tune and Kalrez o-ring. One could postulate that the worn o-ring was allowing leakage into the tube lens skimmer area and the leak was also leaching something out of the o-ring. Normally the o-ring is a total block and any polymers in it would not enter the MS.

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+ 615.7 and 1229.8 Chaps Ions 615.7 Da is [M+H]+ of CHAPS and 1229.8 Da is [2M+H]+ in the sample. Removal can be extremely difficult. Acetone precipitation removes the excess CHAPS, but there may still be an appreciable amount remaining (determined by the above ions in the mass spectrum). CHAPS won't kill your SCX chromatography, but it will chromatograph nicely on reversed phase and you'll get an intense ion at 615 m/z [M+H]+. +136 Series Ions This is exhibited by background (contamination) that has a repeating sequence of 136 amu (TFA sodium salt is 136 Da). Remove TFA from the sample and/or mobile-phase. TFA may be difficult to remove from the column, so take off the column and flow mobile-phase directly into the MS. Identify the source by infusing methanol with a clean syringe and a new piece of tubing. Pentafluoropropionic Acid May stick to PEEK tubing and fittings. This contamination is usually associated with areas that are poorly swept by the flowing liquid (e.g. unions). If you have concerns about the pentafluoropropionic acid, then use fused-silica and steel unions. The ferrules should be kel-F, and PEEK tube nuts should be ok as they do not touch the liquids. Water If any contamination is seen, use high-grade bottled water (Burdick & Jackson HPLC grade.) Avoid ANY nanoPure or MilliQ water! +798 and +803 Contamination Both ions are diisooctyl phthalate analytes derived from plasticizers. Sometimes, their origins can be really hard to track down. If the source is your HPLC, these species can concentrate on your column until the gradient is ramped and the compounds eluted. Check the HPLC solvents by infusion to determine if it's coming from your HPLC or if it's in one of your reagents (e.g., TFA). In the later case, clean the HPLC reservoir bottles. These could be cleaned by rinsing well with 30% Nitric Acid followed, after a water rinse, then by 2M NH4OH. +453.3, +679.5 and +905.7 Contamination Nylon HPLC solvent filters can produce nylon (6,6monomer) peaks at masses of 226 Da, a dimer 452 Da, trimer 678 Da and tetramer 905 Da. The contaminant is very hard to get rid off since it binds very well to C18.

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Peak Clusters at +21, -17, -35, -52 +21 is sodium, -17, -35, -52 are various losses of ammonia and water (if you look at the -17 ion you will probably see a small amount of the -18, as well). Iron-Acetate clusters @ +538, +555 and +534 Mobile-phases with acetic acid can react with iron in the LC/MS system to produce Iron-acetate clusters (as established by Ijames, Dutky, and Fales: J Am Soc Mass Spec 6 1226 1995.) The general formula is Fe3O(CH3CO2)6(L)x where L is a ligand from the mobile-phase (water or organic component, acetonitrile solvent or water. 538 Da could be 6 acetates with no additional ligand. The 555 Da presumably has additional water. 533.88 Da could be a chromium cluster similar to the iron 537.88. Stainless steel in the system contains both iron and chromium. Potential sources in the ESI probe could be the ESI spray needle or the with the metal needle kit. If necessary, try replacing the needle.

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COMMON BACKGROUND IONS FOR ELECTROSPRAY (Positive Ion)

m/z
101 102 104/106 105 120 122 123 130 137 144 145/147 146 150 153 157 159 179 186 214 217 225 231 239/241 242 243 257

Ion
[M+Na]+ [M+H]
+

Analyte
DMSO Triethylamine Acetonitrile Acetonitrile DMSO TRIS Dimethylaminopyridine Diisopropylethylamiine
+

m/z
267 273 279 282 301 317 336 338 360 371 391 413 425 429 445 449 454 522 550 798 803

Ion
[M+H]+ M
+

Analyte
Tributylphosphate Momomethoxytrityl cation Dibutylphthalate Plasticizer in Polyethylene Dibutylphthalate Dibutylphthalate Tributyl tin formate Erucamide
+

[M+Cu]+ [2M+Na]+ [M+Na+CH3CN]+ [M+H]+ [M+H] [M+H]

+ +

[M+H]+

[M+Na]+ [M+K]+ [M+H]

+ +

[M+H]

[M+CAN+NH4] [M+H]+ [2M+Cu]+ [3M+Na]+ [M+H]

+

DMSO TPA Acetonitrile Acetonitrile Phenyldiethylamine 1,8Diazabicyclo[5,4,0]unde c-7-ene(DBU) DMSO Sopdium trifluoroacetate DMSO Tributylamine Unknown Surfactant Unknown Contaminant Dicyclohexyl Urea (DCU) Unknown

[M+Na] [M+H]+ [M+H]+

Erucamide Polysiloxane, followed by 388 Diisooctyl phthalate Diisooctyl phthalate Unknown plasticiser Diisooctyl phthalate Polysiloxane, followed by 462 Dicyclohexyl urea (DCU) Diisooctyl phthalate Unknown Unknown
4 +

[M+Na]+ [M+Na] [M+K]+ [M+H]+ [2M+H]+ [M+Na+ACN]+

+

[M+H]+ [2M+H]+ [M+Na]+ [2M+Na]+ [M+H]

+

[2M+NH ] [2M+Na]+

Diisooctyl phthalate Diisooctyl phthalate

[M+H]+ [M+NH3]+ [(M.HCl)2-Cl] M+ M+ [3M+H]

+ +

PEG polymers exhibit peaks spaced @ 44 amu intervals

Triethylamine Tetrabutylammonium (C4H9)4N+ Trityl cation DMSO

Common Adducts
[M+NH4]+ [M+Na] [M+K]
+ +

[M+18] [M+23] [M+39]

* This information is provided courtesy of New Objective, Inc. 2004

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COMMON FRAGMENT ION AND NEUTRAL FRAGMENTS

Common Small Ions m/z

15 17 18 19 26 27 28 29 30 31 33 34 35(37) 36(38) 39 41 42 43 44 46 56 57 60 79(81) 80(82) 91 127

Composition
CH3 OH H2O H3O, F C2H2, CN C2H3 C2H4, CO, H2CN C2H5, CHO CH2NH2 CH3O SH, CH2F H2S Cl HCl C3H3 C3H5, C2H3N C3H6, C2H2O, C2H4N C3H7, CH3CO C2H4O NO2 C4H8 C4H9 CH4CO2 Br HBr I HI

Common Neutral Fragments m/z loss Composition

1 15 17 18 19 20 27 28 30 31 32 33 33 35(37) 36(38) 42 43 44 45 55 57 59 60 64 79(81) 80(82) 91 127 H CH3 OH H2O F HF C2H3, HCN C2H4, CO CH2O CH3O CH4O, S CH3 + H2O, HS H2S Cl HCl C3H6, C2H2O, C2H4N C3H7, CH3CO CO2, CONH2 C2H5O C4H7 C4H9 C2H3O2 C2H4O2 SO2 Br HBr I HI

301